Correlative Refractive Index Light-sheet Microscopy

Developing future therapies, the first of EPSRC's Healthcare Technologies Grand Challenges, is essential to keep the National Health Service sustainable. Currently, an estimated 70% of the UK's healthcare expenditure goes towards the management of chronic diseases. Regenerative medicine is expected to significantly reduce costs as it can turn chronic, degenerative, diseases into curable conditions. Realising this potential requires the right tools to study the biological development process as it progresses from the single cell to the complex structure of entire organs.

The invention of the optical microscope, and in particular the phase contrast microscope, made it possible to highlight the fine features of living cells with unprecedented clarity. However, cells isolated on a microscope slide often do not behave as tissue in its natural, three-dimensional, environment. The recent development of the planar illumination light-sheet microscope enabled the visualisation of the intact, fluorescently-labelled, organisms during development. While light-sheet microscopy is highly successful for transparent zebrafish or chemically cleared tissue, many tissues are too opaque to be studied beyond the first layer of cells. A prime example is the chick embryo in its early stages of development. The complex collective behaviour of the cells in the initial layer can be studied in exquisite detail, yet as soon as the primitive streak forms, to grow the embryo in the third dimension, we lack the tools to keep track of the cell migration and differentiation. The images are too blurred.

Correlative refractive index light-sheet microscopy aims to make the invisible visible. It is based on the realization that the turbidity of biological samples is due to the same refractive index variations that yield structural information in phase-contrast microscopy. The distribution of optical properties within the specimen not only contains valuable structural information for the biologist, it also enables the adaptive wavefront correction needed for high resolution fluorescence imaging. By developing a hybrid instrument that maps the optical property distribution in parallel with the fluorescence image, this proposal will enable high-resolution deep-tissue imaging in turbid biological specimen. A direct view into the inner workings of the biological development process is essential to develop effective regenerative-medicine therapies.

The immediate impact of this project will undoubtedly be felt in biology and in particular developmental biology where progress in the state-of-the-art in microscopy directly dictates the boundaries of knowledge. Knowledge that is of strategic importance for the development of effective therapies through regenerative medicine and crucial to reduce the financial pressures on the National Health Service. A microscope that can image large turbid samples with high contrast and resolution will break through the imaging depth barrier in biological samples. To facilitate adoption, the prototype instrument must however be easy to use and readily available to biology research groups and biotech or pharmaceutical companies. I therefore hope to leverage my ties with industry and employ my experience in commercializing imaging solutions to the outcomes of the grant. In particular life science instrumentation companies with an established market and contacts in the biomedical and pharmaceutical research will be able to bring the novel technology to market on the shortest possible time scale. To achieve this, M Squared Lasers Ltd is involved from the outset with this Fellowship.

Furthermore, this research project will yield much information about the optical properties of such samples. At present our knowledge of the optical properties within thick biological specimen is limited. It is anticipated that a tool to measure the optical properties of large specimen in detail will lead to a broader range of studies and greater insights into the diversity of the optical property distributions between species and their variation during development. This places us in a privileged position to direct the development of the next generation of microscope technology here in the UK.